The recombinant vaccine containing Aventis Pasteur's Canarypox vector clade A/B/E (ALVAC)-HIV and gp120 alum, first pioneered 20 years ago, decreased the risk of HIV acquisition by 31.2% in the RV144 HIV vaccine trial in Thailand, and it did so despite negligible CD8+ cytotoxic T cells and neutralizing antibody titers against primary HIV strains. The result of this trial defied expectations in the field that it would fail since this vaccine platform was not considered sufficiently immunogenic. Some investigators have been invigorated by this unexpected positive result, as RV144 may indicate that a vaccine for HIV may be feasible through the elicitation of non-canonical immune responses. Others continue to believe that the key to HIV prevention lies in preventive antiretroviral therapy or the life-long administration of broadly neutralizing antibodies. HIV is a formidable pathogen that has evolved a variety of mechanisms to hijack every arm of the human immune system. Given that variables as diverse as population genetics, nutrition, age, sex, and co-infecting agents might affect the efficacy of any intervention, the development of different approaches is a strategic necessity to prevent infection and eradicate this virus. Having confirmed the results of RV144 and adapted them into the macaque model, our work has identified a novel correlate in monocyte innate memory responses that involves the metabolic and epigenetic reprogramming of myeloid cells. This response is persistent and it has been shown to be effective against infectious agents and some forms of cancer. In addition, our work has uniquely demonstrated several correlates of both decreased and increased risk of SIVmac251 acquisition, suggesting mechanistic insights and possible remedies. Increased risk of SIVmac251 acquisition: Cellular immunity: Activated gut-homing CD4+ T cells, mucosal IFN-? producing NKG2Anegative NKp44negative cells, and CD16+ monocytes. Cytokines: Plasma IL-6 and IL-8 in old macaques. Humoral: Antibodies to V1. Systems biology: STAT3 activation. Decreased risk of SIVmac251 acquisition: Cellular immunity: CD14+ monocytes (innate memory), gut-homing CCR5negative Th2 cells, and gut mucosal NKp44+ cells. Humoral: Antibodies to V2. Systems biology: RAS, inflammasome, and hypoxia. We have gained mechanistic insights into the efficacy of the DNA/ALVAC/gp120/alum vaccine regimen (see accompanying manuscript by Vaccari et al., Nat Med 2018). Firstly, the intramuscular priming with the Gag and Envelope DNAs (a productive collaboration with Dr. Felber and Dr. Pavlakis) and boosting with ALVAC-SIV, both able to express SIV pseudo-virions, focuses the antibody response to conformational V2 epitopes. Secondly, the DNA prime collaborates with the ALVAC and alum adjuvants to activate the inflammasome and release IL-1b in CD14+ cells. Consistent with the model proposed in, ALVAC has a tropism for CD14+ monocytes and its cytosolic DNA activates the inflammasome through the engagement of cGAS, IF116, and AIM2, key components of the inflammasome. We have shown that within 24 hours of immunization, ALVAC causes more than 50-fold higher plasma levels of IL-1b, a cytokine tightly regulated by inflammasome activation, than do the Ad26, MVA, or NYVAC primes. Similarly, the DNA vaccine activates cGAS IF116 via cross-presentation. AIM2 knockout mice experiments demonstrated the essential role of the inflammasome in DNA vaccination and alum alhydrogel is likewise a strong activator of the inflammasome. IL-1b expression is associated with emergency myelopoiesis and induces differentiation of hematopoietic stem and progenitor cells via the PU.1 transcription program. Indeed, we have found a correlation with CXCR4+ premonocytes (cells recently released from the bone marrow) and a decreased risk of virus acquisition as well as a decrease in the number of transmitted virus variants in the animals that become infected. However, our data suggest that while necessary, the early IL-1b burst may need to be short-lived to avoid the accumulation of an excessive level of pro-inflammatory macrophages in tissues. The engagement of CD14+ monocytes suggests a role for trained immunity, a well-characterized mechanism of BCG-induced innate memory immunity, that can be recapitulated in vitro by b-glucan stimulation of the inflammasome in CD14+ monocytes, aerobic glycolysis, and epigenetic reprogramming through the HIF-1? and mTOR pathways. Systems biology in our study demonstrated a correlation of both inflammasome activation and hypoxia with delayed SIVmac251 acquisition. The trained monocytes' epigenome change can be long-lasting and, upon b-glucan restimulation, monocytes respond with heightened production of pro-inflammatory TNF-?, IL-6, and IL-8 cytokines (trained cells) or anti-inflammatory IL-10 and TGF-b cytokines (tolerant cells), depending on the microenvironment and dose and type of the stimulus. Interestingly, we found that ALVAC immunization induces a burst of not only IL-1b, but also IL-10 within 24 hours of vaccination in vivo. IL-10 is also induced by alum and may be produced by neutrophils that are increased by ALVAC vaccination. By inhibiting the inflammasome and mTOR pathways and IL-1b release, IL-10 may stir monocyte memory toward a more tolerant state. This hypothesis is supported by the finding in vaccinated macaques of high levels of miRNAs in extracellular vesicles that target mTOR, IL-6, and the inflammasome. Interestingly, IL-10 has been demonstrated to reduce CCR5 expression on CD4+ T cells and, accordingly, we found that the Th2 cells that correlate with a decreased risk of viral acquisition do not express CCR5. Thus, we hypothesize that IL-10 may reprogram monocyte memory toward a tolerant state and decrease T cell activation. Our previous finding that the RAS pathway correlates with protection in the ALVAC prime as well as in the gp96 prime regimens can be integrated with the current findings because of the essential role of the MAPK/ERK pathway in licensing function of the inflammasome post-translationally and the ability of hypoxia to activate the inflammasome by increasing the levels of NLRP3, AIM2, and pro-IL-1b in monocytes. Our finding that age affects the efficacy of the DNA-ALVAC-SIV-alum vaccine regimen further supports the role of monocytes in vaccine efficacy as it is well documented that the functionality of monocytes declines with aging. Thus, the difference in challenge outcomes in young and old animals provides a means to contrast immunological, gene expression, and epigenetic parameters and may guide us to rationally design adjuvants able to overcome the age bias and increase the durability of vaccine efficacy. Collectively, our data indicate that vaccine platforms able to tip the balance toward the generation of more target cells and/or durable innate inflammatory responses are not protective, even if they induce high levels of anti-V2 antibodies. Instead, vaccines able to induce anti-V2 (but not V1) antibodies and CD14+ cells linked to Th2 CCR5negative CD4+ T cells decrease the risk of SIV acquisition. Production of IL-10 and STAT3 activation may likewise be beneficial in curbing inflammatory response, but if excessive, it may cause an immune suppressive environment that also affects protective innate and adaptive responses and negates vaccine efficacy. In the coming years, we plan to study the intersection of innate responses and the role of interfering vaccine-induced antibodies in antiviral controls and possibly develop a vaccine platform effective for all ages.